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Metaphase chromatids are believed to consist of loops of chromatin anchored to a central scaffold, of which a major component is the decatenatory enzyme DNA topoisomerase II. Silver impregnation selectively stains an axial element of metaphase and anaphase chromatids; but we find that in earlier stages of mitosis, silver staining reveals an initially single, folded midline structure, which separates at prometaphase to form two chromatid axes. Inhibition of topoisomerase II prevents this separation, and also prevents the contraction of chromatids that occurs when metaphase is arrested. Immunolocalization of topoisomerase II alpha reveals chromatid cores analogous to those seen with silver staining. We conclude that the chromatid cores in early mitosis form a single structure, constrained by DNA catenations, which must separate before metaphase chromatids can be resolved.

Mitotic chromosome condensation is normally dependent on the previous completion of replication. Caffeine spectacularly deranges cell cycle controls after DNA polymerase inhibition or DNA damage; it induces the condensation, in cells that have not completed replication, of fragmented nuclear structures, analogous to the S-phase prematurely condensed chromosomes seen when replicating cells are fused with mitotic cells. Caffeine has been reported to induce S-phase condensation in cells where replication is arrested, by accelerating cell cycle progression as well as by uncoupling it from replication; for, in BHK or CHO hamster cells arrested in early S-phase and given caffeine, condensed chromosomes appear well before the normal time at which mitosis occurs in cells released from arrest. However, we have found that this apparent acceleration depends on the technique of synchrony and cell line employed. In other cells, and in synchronized hamster cells where the cycle has not been subjected to prolonged continual arrest, condensation in replication-arrested cells given caffeine occurs at the same time as normal mitosis in parallel populations where replication is allowed to proceed. This caffeine-induced condensation is therefore "premature" with respect to the chromatin structure of the S-phase nucleus, but not with respect to the timing of the normal cycle. Caffeine in replication-arrested cells thus overcomes the restriction on the formation of mitotic condensing factors that is normally imposed during DNA replication, but does not accelerate the timing of condensation unless cycle controls have previously been disturbed by synchronization procedures.